One of the major questions in biology is how the one dimensional information encoded in an organism's DNA directs the four dimensional pattern of events that leads to the complex developmental changes that occur during its life cycle. Elucidation of any aspect of this basic question gained from studying specific well-defined developmental systems, should be of significant benefit for gaining a better understanding of normal, and abnormal, development in all eukaryotes. The genetic control of sex determination in Drosophila melanogaster is an attractive system in which to study this broad question at a specific level, since several of the key genes controlling sexual differentiation have been identified by genetic analyses, and some of these have been isolated by molecular cloning. Studies done to date have given us insight into how these genes interact with one another as parts of a single regulatory hierarchy controlling sexual development. This proposal focuses on the continued genetic and molecular characterization of one of these developmentally important regulatory genes, the transformer (tra) gene. In vitro mutagenesis followed by P-element transformation methods will be used to define sequences important for the sex-specific splicing of the tra pre- mRNA. Anti-tra antibodies will be generated using oligopeptides or fusion proteins as immunogens, and these will be used to examine the interaction of the tra gene product with other cellular constituants such as the tra-2 and dsx gene products. These antibodies will also be used to examine the tissue- and sex-specific patterns of expression of the tra gene's (presumed) polypeptide product(s). A large scale mutagenesis experiment will be carried out in an attempt to isolate genes that play a role in regulating tra gene expression. DNA sequence analyses, coupled with other molecular biology methods (e.g. RNA blot hybridization, RNase protection assays, p-element transformation, etc.) will be used to investigate aspects of the tra gene's evolution, including its mechanism of sex-specific regulation (alternative splicing).